Steritop filter unit

Metanephros organ culture was performed as we described previously [11 ]. Briefly, SD female rats of known mating date were anesthetized and laparotomized. Fetuses were aseptically removed, and metanephroi from fetuses at embryonic day 14 (E14) were collected and freed of exogenous tissue. Explants were placed onto a Steritop filter unit (Millipore, Billerica, MA, USA) floating on a defined serum-free medium and incubated for 6 d in 35 mm Petri dishes at 37°C in a humidified incubator (5% CO₂). The defined medium was composed of Eagle's Minimum Essential Medium containing 10% (v/v) fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. All of these reagents were obtained from Sigma (St. Louis, MO, USA). The culture medium was changed daily, and no antibiotic or fungicide was present throughout the experiment. Fresh aliquots of each culture medium additive were used for each metanephros culture. The medium was changed daily. Metanephroi were treated with melatonin (1 μM and 1 mM) and harvested after 6 d for real-time polymerase chain reaction.

Streptococcus mutans was cultured in BHI broth for 24 h at 37°C with 5% CO₂. After overnight growth, 1 mL of a standardized suspension containing 10⁷ cells/mL was inoculated into 6 mL of BHI broth and incubated for 4 h at 37°C (5% CO₂) (Barbosa et al., 2016 (link)). The culture was centrifuged at 5000 rpm for 10 min and the supernatant was filtered with a 0.22 μm diameter pore membrane using a vacuum filtration system (Stericup^® and Steritop^® Filter Unit, Millipore, MA, United States).

Monitoring of the ¹³CO₂ level in air expired by mice was performed as described previously23 (link) with some modifications. The apparatus was composed of a bottle top filter unit (animal chamber; Steritop filter units, Millipore, Billerica, MA), peristaltic pump (Masterflex, Cole-Palmer Instruments Co., Vernon Hills, IL) and a breath-sampling bag (Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan). The animal chamber was wide enough to allow the mouse to turn around. Mice were fasted for 18 h and had free access to water. Racol containing [¹³C]-labeled acetic acid (16 mg acetic acid/kg, 5 ml racol/kg) was then intragastrically administered. Expired air containing ¹³CO₂ was collected 5, 10, 15, 20, 25, 30, 40, 50, 60 and 90 min after administration of [¹³C]-labeled acetic acid. The ventilation volume was 50 ml min⁻¹. The content of ¹³CO₂ in collected expired air was measured with a POC-one analyzer (Otsuka Electronics Co. Ltd., Osaka, Japan). Values are presented as the difference of ¹³CO₂/¹²CO₂ (Δ¹³CO₂ (%)) between each sample and the standard air. For evaluation of the gastric emptying, we determined the half-time for gastric emptying (T_1/2).